Metal homeostasis and resistance in bacteria

Abstract

Metal ions are essential for many reactions, but excess metals can be toxic. In bacteria, metal limitation activates pathways that are involved in the import and mobilization of metals, whereas excess metals induce efflux and storage. In this Review, we highlight recent insights into metal homeostasis, including protein-based and RNA-based sensors that interact directly with metals or metal-containing cofactors. The resulting transcriptional response to metal stress takes place in a stepwise manner and is reinforced by post-transcriptional regulatory systems. Metal limitation and intoxication by the host are evolutionarily ancient strategies for limiting bacterial growth. The details of the resulting growth restriction are beginning to be understood and seem to be organism-specific.

Figure 1. Types of metalloregulatory systems

Metal sensing regulators can be divided into three classes: proteins that bind metal directly, proteins that bind a metal dependent cofactor, and riboswitches that bind metal directly. (A) Direct metal sensors are those which modulate transcription in response to direct metal binding (for example, Zn(II) binding to Zur). (B) Product sensing metalloregulators use the levels of a metal dependent metabolite as a proxy for intracellular metal levels. In the case of B. japonicum Irr, heme serves as a proxy for Fe(II) levels. Irr binds directly to ferrochelatase, which catalyzes formation of heme through the insertion of iron into protoporphyrin. Under conditions of Fe(II) sufficiency, heme is produced by ferrochelatase. Heme can then bind Irr, leading to its degradation. However, under conditions of Fe(II) limitation, apo-Irr is released and active for transcription regulation. (C) Metal sensing riboswitches, such as the yybP-ykoY Mn(II) sensing riboswitch, can act at the level of transcription and translation^,. In B. subtilis, binding of Mn(II) favors a RNA conformation that prevents the formation of an intrinsic transcription termination hairpin.

Figure 2. Mechanisms of stepwise regulation of the Zur regulon

Under conditions of Zn(II) sufficiency (right), the dimeric Zur repressor is present in its fully metallated (Zur₂:Zn₄) state and the full Zur regulon is repressed. As Zn(II) levels fall, the first sets of genes (see Fig. 3) are derepressed as Zur transitions to the intermediate metallated form (Zur₂:Zn₃; middle) that binds DNA with lower affinity. As Zn(II) levels fall further, the remaining, more tightly bound, Zn(II) ion dissociates leading to formation of Zur with only the structural Zn(II) site is occupied ( Zur₂:Zn₂; left). This leads to derepression of additional adaptive responses, including the expression of the alternate folate synthesis enzyme FolEB and an alternate S14 ribosomal protein.

Figure 3. Metalloregulation in B. subtilis as a model system

As cells transition from Zn(II) sufficiency to Zn(II) deficiency, the Zur regulon is derepressed in three stages. First, Zn(II) independent L31 and L33 ribosomal protein paralogs are expressed to liberate Zn(II) from the ribosome. Then, the ZnuABC Zn(II) uptake system is expressed to import Zn(II) from the environment. Finally, the Zn(II)-independent S14 ribosomal protein paralog is expressed to ensure continued synthesis of ribosomes, and the Zn(II)-independent. FolEB GTP cyclohydrolase is produced to support folate synthesis. Under conditions of Zn(II) excess, expression of Zn(II) efflux pumps is derepressed when Zn(II) binds to CzrA, which impairs its ability to bind to its operator sites. Whereas conditions of Zn(II) limitation and excess are sensed by two metalloregulators, Mn(II) and Fe(II) homeostasis are controlled by single metalloregulatory proteins (Mn(II) by MntR and Fe(II) by Fur). Under conditions of Mn(II) limitation, the MntR regulon is derepressed leading to expression of two Mn(II) uptake systems. When cells are exposed to Mn(II) excess, MntR directly activates the expression of Mn(II) efflux pumps. As cells become severely Mn(II) overloaded, genes regulated by the Mn(II) sensing yybP-ykoY riboswitch are induced^,. Fe(II) limitation leads to derepression of the Fur regulon, including genes required for Fe(II) uptake (siderophore biosynthesis and uptake, elemental iron import, and an iron-citrate importer) and the Fe(II)-sparing response. Under conditions of Fe(II) intoxication, Fur directly induces expression of Fe(II) efflux mediated by PfeT .

Figure 4

Metal homeostasis during bacteria-host interactions and the evolutionary origins of innate immunity. (see Box 4 text).